The fundamental objective of the proposed research is to investigate the role of non-histone proteins in maintaining the structure of the HeLa genome through the cell cycle. To this end, polyacrylamide gel electrophoresis will be applied to compare in a quantitative manner the non-histone proteins that are associated with HeLa metaphase chromosomes, nuclei and chromatin preparations. In addition, two-dimensional gels (isoelectric focusing/SDS discontinuous) will be applied to characterize the non-histone proteins to a high resolution. With these two-dimensional gels, the synthesis and modifications (phosphorylations, acetylations, poly ADP-ribosylations, etc.) or the HeLa proteins of synchronized cells will be followed through the cell cycle. The major non-histone proteins which are implicated in maintaining the long-range organization of the HeLa genome will be purified and further characterized by various biochemical and physical chemical techniques. As part of this proposed research, the histone-depletion procedure will be extended to other stages of the cell cycle. Preliminary results have suggested that non-histone proteins help to maintain the organization of HeLa DNA in interphase nuclei. In addition, nuclei from defined stages of the cell cycle will be treated with nucleases besides extracting the histones to produce structures analogous to the metaphase "scaffolds". Electron microscopy will be used to learn something of the organization of the DNA in histone-depleted nuclei.